There is mounting evidence that an organization of Krebs TCA cycle and associated enzymes exists within the mitochondrial matrix. It was recently reported that the metabolism of [2-13C] or [3-13C] propionate in yeast gave rise to asymmetrically labeled alanine. This observation has at least two explanations. It is possible that an oxidative pathway with no symmetrical intermediates exists for the direct conversion of propionate to pyruvate. Such a pathway has been described in some organisms, but not Saccharomyces cerevisiae. Alternatively, if the Krebs TCA cycle enzymes are highly organized, then one could visualize an orientation conserved transfer of intermediates between succinate thiokinase, succinate dehydrogenase, and fumarase to explain this somewhat unexpected result. To test whether the intermediates succinate and fumarate are transferred from one active site to another without their release to a bulk phase, we examined the metabolism of [4-13C] glutamate in Saccharomyces cerevisiae. The [13C] aspartate formed showed a [2-13C]/[3-13C] ratio greater than 1. When [2-13C] acetate is incubated with yeast the C2/C3 aspartate ratio is 1.5 at 2 min and decreases to 1.0 after about 10 minute of incubation. These results indicate that orientation conserved transfer occurs at the succinate thiokinase and succinate dehydrogenase steps of the Krebs cycle. We have also examined the metabolism of yeast cells transformed with a fusion protein of citrate synthase and malate dehydrogenase. Using 13C NMR we showed that these transformed cells carried out orientation conserved transfer efficiently. No changes in the general metabolic pattern of these cells occurred. On the other hand a control cell line containing a citrate synthase-serum albumin fusion protein showed a loss of orientation conserved transfer.